Radiopharmaceutical chelates and method of external imaging

ABSTRACT

A chelate of technetium-99m, cobalt-57, gallium-67, gallium-68, indium-111 or indium-113m and substituted iminodiacetic acid or an 8-hydroxyquinoline useful as a radiopharmaceutical external imaging agent. The invention also includes preparative methods therefor.

BACKGROUND OF THE INVENTION

This is a continuation-in-part application of our copending U.S.application Ser. No. 555,037, filed on Mar. 3, 1975, now abandoned.

Radiopharmaceutical imaging agents have been utilized heretofore for theexternal imaging of various portions of the anatomy. Onlyradiopharmaceuticals which emit gamma-photons are suitable for thisutility. The field of application is restricted due to the fact that ofthe radionuclides which emit gamma rays, very few meet the additionalrequirements imposed by the inherent limitations of exiting imagingsystems and by the necessity of keeping the radiation dose as low aspossible. Among these requirements are the need for a simple gammaspectrum, a high yield of photons having an energy sufficiently low topermit effective collimation and efficient detection and a half-lifesufficiently short to permit the admininstration of millicuriequantities without an excessive post-test radiation dose.

The usual method of external imaging generally comprises labeling ortagging an organic compound suitable for administration to a patientwith a suitable radio-isotope. More particularly, a biological agentknown to localize in the particular organ or anatomical section to beimaged is labeled to a small extent with a radio-isotope. The thuslabeled biological agent then permits external imaging of the desiredorgan utilizing conventional radio scanning techniques.

The problems associated with prior art attempts in this direction centermainly on combining the requirements (1) that the biological agent bespecific to the organ to be imaged (2) that a suitable radionuclide beemployed as the labeling agent (3) that the labeled agent issufficiently stable in vivo to permit effective imaging and (4) that thelabeled biological agent retains its organ specificity.

It is an object of the present invention to provide a radiolabeledbiological agent having a high degree of in vivo stability and which ishighly organselective. It is a further object of the invention toprovide a method of external imaging employing said agent. It is still afurther object of the invention to provide a method for the preparationof said agent.

SUMMARY OF THE INVENTION

The above objects are achieved by providing a radiolabeled diagnosticagent which combines the high target organ specificity of various drugsand biochemicals with the excellent nuclear imaging properties of theradiometals technetium-99m, cobalt-57, gallium-67, gallium-68,indium-111 or indium-113m.

The invention is predicated on the discovery that chelates of the aboveradiometals with a substituted iminodiacetic acid or an8-hydroxyquinoline have a high degree of in vivo stability, are highlyspecfic to certain organs or anatomical sections and posses excellentnuclear imaging properties.

The above chelates may be prepared by reacting the desired radio-isotopewith the chelating agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing in vivo distribution of a product according tothe invention.

FIG. 2 is a graph showing in vivo distribution of another productaccording to the invention.

FIG. 3 is an anterior imaging study, after injection of a productaccording to the invention.

FIG. 4 is an anterior imaging study at a later time than FIG. 3.

FIG. 5 is an imaging study of a Rose Bengal product vs. a productaccording to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Technetium-99m is commercially available either from an isotopegenerator as a daughter product of molybdenum-99 or as a direct productfrom a commercial supplier. It is also available as a solvent extractionproduct from molybdenum-99 solutions generally as alkali metalpertechnetate solutions at 5-100 mCi. A further discussion ofpreparative methods appears in U.S. Pat. Nos. 3,468,808 and 3,382,152.

The technetium-99m chelate is most preferably prepared by reducing asolution of a pertechnetate, e.g., an alkali metal pertechnetate in thepresence of the chelating agent. The reduction is preferably effectedutilizing stannous chloride as a reducing agent. Any suitable reducingagent may be employed including other stannous salts such as stannouspyrophosphate. As a result of this reduction step, the product will alsocontain a significant proportion of the stannous chelate. It is to beunderstood that the present invention includes the product mixturecontaining both the radiometal chelate and the corresponding stannouschelate.

Indeed, the composition of the invention is most conveniently providedas a sterile kit consisting of non-radioactive chemicals for mixing withthe radiometal source prior to use. The kit preferably contains astannous salt solution, pH buffer solution or combinations thereof.Using sterile reagents and aseptic techniques, the respective solutionswould be mixed with each other in any desired order and then with theradiometal source solution. The resulting solution containing theradiometal chelate, te stannous chelate and any free chelate may then beemployed directly for imaging purposes.

Generally, a solution adapted for intravenous administration containingup to 15 mCi of radioactivity is administered to the patient. Generally,this may be accomplished by administering 0.2-1 ml of a solutioncontaining from about 2 to about 100 mg of combined chelate product.Radioassay of the radio-isotope in the desired organ may be accomplishedutilizing equipment, such as a scintillation camera, etc.

Organ specificity is determined by the particular chelating agentemployed. All of the chelates according to the present invention,however, are cleared through either the kidneys or liver. Therefore, thechelates of the above radiometals with most substituted iminodiaceticacids and 8-hydroxyquinolines may be utilized for the imaging of theseorgans.

Preferably, the chelating agents are of the formulae ##STR1## wherein Rmay be alkyl of up to about 24 carbon atoms preferably about 14 carbonatoms, alkenyl, aryl alkyl or cyclo-aliphatic groups substituted withhalogen, hydroxy, carboxy, nitro, amino, keto or heterocyclic groups.The groups may be interrupted by ether or thio-ether linkages.

The most preferred chelating agents are the substituted iminodiaceticacid and 8-hydroxyquinoline analogs of drugs and biochemicals whoseorgan specificity characteristics are known.

Other specific chelating agents suitable for use in the practice of theinvention are N-methyl-iminodiacetic acid, N-(10-carboxydecyl)iminodiacetic acid, N-[N'-(2,6-dimethylphenyl) carbamoylmethyl]iminodiacetic acid, N-(o-bromobenzyl) iminodiacetic acid,N-[3-(1-naphthyloxy)-2-hydroxypropyl] iminodiacetic acid,nitrilotriacetic acid, or 5,7-diiodo-8-hydroxyquinoline.

It is to be understood that the term "substituted iminodiacetic acid" isintended to include those compounds wherein R in the above structuralformula combines with each methylene group to form a heterocyclic ring.An example of such an acid is 2,6-pyridinedicarboxylic acid.

The gallium and indium chelates ae prepared by the addition of eitherGaCl₃ or indium chloride in 0.05 M HCl to the appropriate chelatingagent at pH 3.5. After a 25-minute incubation period, the pH is raisedto between 5 and 7.

The invention is illustrated by the following non-limiting examples.

EXAMPLE 1

2 grams (0.01 moles) of alpha-chloro-2,6-acetylxylidine and 2 grams(0.01 moles) of iminodiacetic acid (disodium salt) were refluxed in 200ml of a 3:1 ETOH/H₂ O mixture for 48 hours. The mixture was evaporatedto dryness to yield a yellow residue. 25 ml of H₂ O were added to theresidue. That which failed to go into solution was collected by vacuumfiltration. To the filtrate concentrated hydrochloric acid was addeddrop-wise and the pH monitored. At pH 3 the clear solution became cloudyand was cooled overnight. An off-white precipitate was collected whichwas recrystallized from boiling water. The product was identified asN-[N'-(2,6-dimethylphenyl) carbamolymethyl] iminodiacetic acid. m.p.201°-203°. Percent yield 20% of theoretical.

    ______________________________________                                        NMR:     DMSO-d.sub.6                                                                            δ = 7.11 (s,3, aromatic protons)                                        δ = 3.63 (s,4, CH.sub.2 --COO--)                                        δ = 3.57 (s,2, --CH.sub.2 --N<)                                         δ = 2.20 (s,6, CH.sub.3)                             CHN:     57.13 C 6.16 H 9.52 N Theor                                                   57.10 C 6.23 H 9.43 N Exp                                            ______________________________________                                    

EXAMPLE 2

The N-[N'-(2,6-dimethylphenyl) carbamoylmethyl] iminodiacetic acidprepared according to Example 1 in an amount of 150 mg (0.51 mmoles) wasdissolved in 3 ml of 0.1 N NaOH. The pH of the solution was adjusted to3.5 with 1 N HCl. Extra 0.1 N NaOH was added thereto to compensate forthe acidic SnCl₂ solution which follows. 0.3 cc of a solution of SnCl₂(20 mg. 0.11 mmole in 10 ml of 1 N HCl) was added. After a five-minutewait 80 microcuries of technetium-99m as sodium pertechnetate was added.The product was chromatographed in saline and recorded on aradiochromatogram scanner. The resulting graph showed a peak at thesolvent front, R_(f) ═1 due to the chelated compound. There was littlecolloid formation. There was substantially no free technetium-99m(TR_(f) ═75).

EXAMPLE 3

Methyl iminodiacetic acid in an amount of 150 mg was dissolved in 3 mlof 0.1 N NaOH. The pH of the solution was adjusted to 3.5 with 1 N HCl.Extra 0.1 N NaOH was added thereto to compensate for the acidic SnCl₂solution which follows. 0.3 cc of a solution of SnCl₂ (20 mg. 0.11 mmolein 10 ml of 1 N HCl) was added. After a five-minute wait 80 microcuriesof technetium-99m as sodium pertechnetate was added. The product waschromatographed in saline and recorded on a radiochromatogram scanner.The resulting graph showed a peak at the solvent front, R_(f) ═1 due tothe chelated compound. There was little colloid formation. There wassubstantially no free technetium-99m (TR_(f) ═0.75).

EXAMPLE 4

2 μ Ci(technetium-99m) of the product of Example 2 were injectedintravenously into mice. The animals were sacrificed serially afterinjection and the activities in major organs were determined by countingmultiple samples from each organ in a scintillation counter. The in vivodistribution of the product of Example 2 in the mice were plotted as afunction of time as shown in FIG. 1.

EXAMPLE 5

The procedure of Example 4 was followed utilizing the product of Example3. The in vivo distribution of the product in mice as a function of timewere plotted as shown in FIG. 2.

EXAMPLE 6

4 mCi (technetium-99m) of the product of Example 2 were intravenouslyinjected into laboratory dogs. One animal was selected for imaging atvarious time intervals utilizing a scintillation camera. Camera imageswere obtained in multiple exposures and demonstrated the localization oftechnetium-99m in the liver. See FIG. 3, which depicts anterior imagingstudies and demonstrates the rapid uptake by the liver which is clearlyidentified at 5 minutes. (Frame A). The gall bladder appears as a colddefect. Sequential images taken at 25, 40 and 50 minutes are shown inFrames B, C, and D, in which clearance from the liver is demonstratedwith progressive accumulation of the radiopharmaceutical in the gallbladder. Less than 10% and 3% of the injected dose remained in the bloodat 10 minutes, respectively. Sufficiet cholecystokinin was injected intothe dog intravenously to effect contraction of the gall bladder.Sequential studies revealed radiopharmaceutical activity progressingthrough the small intestines, seen in FIG. 4. Within 1 minute of theinjection of cholecystokinin the technetium-99m labeled product is seenleaving the gall bladder (Frame E). Frames F, G and H taken at 5, 10 and35 minutes show a bolus of activity moving progressively through a smallintestine. The images were obtained using a gamma scintillation camera(Pho Gamma III) and a parallel hole high sensitivity collimator.

EXAMPLE 7

The procedure of Example 6 was carried out and the results compared withthose obtained following injection of the same dog at a later time withI-131 Rose Bengal. Both before and after plasma loading withbromosulphthalein (BSP) to simulate hyperbilirubinemia, BSP levels of4-7 mg percent did not substantially alter the plasma clearance orimaging characteristics of the techmetium-99m labeled product. Theseimages were of much better quality when compared to those obtainedsubsequently in the same dog using I-131 Rose Bengal, as shown in FIG.5.

EXAMPLE 8

The procedure of Examples 2 and 3 was followed to prepare thetechnetium-99m chelate of 8-hydroxyquinoline, employing a 7 m-molarsolution of 8-hydroxyquinoline and an acidic stannous chloride reducingsolution. The chelate was recovered by chloroform extraction at a yieldgreater than 90%.

Biodistribution studies were undertaken utilizing the procedure ofExample 4. 2μ Ci (technetium-99m) of the above chelate were injectedintravenously into 25 g mice. The animals were sacrificed after 60minutes and the activities in major organs were determined by countingmultiple samples from each organ in a scintillation counter. It wasdetermined that on an average, 40% of the injected dose appeared in theliver and 20% in the intestines.

EXAMPLE 9

The gallium-67 chelate of 8-hydroxyquinoline was prepared by adding Ga⁶⁷Cl₃ in 0.05 M HCl to an aqueous 7 m-molar 8-hydroxyquinoline solutionhaving a pH of 3.5. Following a 25 minute incubation period the pH israised to 6. Chloroform extraction of the reaction product produceda >90% yield of the chelate. Biodistribution studies were undertakenaccording to the procedure outlined in Example 8. Following intravenousinjection of the chelate into 25 g mice, 25% of the injected dose wasfound in the liver, 13% in the intestines and 20% in the blood after 60minutes.

EXAMPLE 10

The technetium-99m chelate of nitrilotriacetic acid was preparedaccording to the stannous chloride reduction method outlined in Examples2, 3 and 8. The chelate is water-soluble with >95% migration in salineemploying paper chromatography. Biodistribution studies were carried outaccording to the procedure outlined in Example 8. The chelate was foundto rapidly clear through the kidneys to urine (40% eliminated in urineafter 60 minutes) with less than 5% of the injected dose found in theliver and intestines.

EXAMPLE 11

The cobalt-57 chelate of N-[N'-(2,6-dimethylphenyl) carbamoylmethyl]iminodiacetic acid was prepared by heating 2-5μ Ci of Co⁵⁷ Cl₂ in thepresence of 1 ml (20 mg/ml) of a solution of the compound (pH 4-5) for 1hour at 100° C. The chelate was chromatographed and biodistributionstudies carried out using the procedure of Example 8. At 30 minutes, 28%of the injected dose appears in the liver and 12% in the intestines.

EXAMPLE 12

The technetium-99m chelate of 10-carboxydecyliminodiacetic acid wasprepared according to the stannous chloride reduction method of Examples2, 3 and 8. The product was chromatographed in saline. >98% the materialhad an R_(f) ═1. Biodistribution studies of the chelate according toExample 8 in ten 25 g mice showed rapid blood clearance with less than6% of the injected dose remaining in the blood at 60 minutes.Radioactivity was eliminated through both kidneys and liver withpersistent activity noted in the liver and lungs.

EXAMPLE 13

The technetium-99m chelate of N-(o-bromobenzyl) iminodiacetic acid wasprepared by the stannous chloride reduction method described in Examples2, 3 and 8. The product was paper chromatographed in saline (98% had anR═1.) Biodistribution studies carried out on twelve 25 g mice accordingto the procedure of Example 8 showed rapid blood clearance (less than 5%remainig at 60 minutes) with a high uptake in the liver (40%) andintestines (30%) at 30 minutes.

EXAMPLE 14

The procedure of Example 11 was followed to prepare the cobalt-57chelate of methyliminodiacetic acid.

EXAMLPE 15

The procedure of Example 9 was followed to prepare the gallium-67chelate of methyliminodiacetic acid. Biodistribution studies carried outaccording to the procedure of Example 8 showed rapid renal clearance.

EXAMPLE 16

The stannous chloride reduction procedure of Examples 2, 3 and 8 wasemployed to prepare the technetium-99m chelate of5,7-diiodo-8-hydroxyquinoline.

EXAMPLE 17

The stannous chloride reduction method of Examples 2, 3 and 8 was usedto prepare the technetium-99m chelate of 2,6-pyridinedicarboxylic acid.

We claim:
 1. A chelate of technetium-99m, cobalt-57, gallium-67,gallium-68, indium-111 or indium-113m and a substituted iminodiaceticacid .[...]..Iadd., said chelate, upon intravenous administration, beingliver and/or gallbladder selective..Iaddend. .[.2. A chelate oftechnetium-99m cobalt-57, gallium-67, gallium-68, indium-111 orindium-113m and an 8-hydroxyquinoline..].
 3. A composition comprising amixture of the technetium-99m chelate of claim 1 and the stannouschelate of said chelating agent.
 4. A composition comprising a mixtureof the technetium-99m chelate of claim 1, the stannous chelate of saidchelating agent and said chelating agent.
 5. The chelate of claim 1wherein said iminodiacetic acid chelating agent is N-methyliminodiaceticacid, N-[N'-(2,6-dimethylphenyl) carbamoylmethyl] iminodiacetic acid,N-(10-carboxydecyl) iminodiacetic acid, N-(O-bromobenzyl) iminodiaceticacid, N-[3-(1-naphthyloxy)-2-hydroxypropyl] iminodiacetic acid,.[.nitrilo-triacetic acid.]. or 2,6-pyridinedicarboxylic acid. 6.N-[N'(2,6-dimethylphenyl) carbamoylmethyl] iminodiacetic acid.
 7. Amethod of external imaging which includes the intravenous administrationof a solution adapted for intravenous administration containing thechelate of claim
 1. .[.8. A method of external imaging which includesthe intravenous administration of a solution adapted for intravenousadministration containing the chelate of claim 2..].
 9. A method ofpreparing the chelate of claim 1 comprising reacting said radio-isotopewith said chelating agent.
 10. The method of claim 9 wherein saidradioisotope is technetium-99m.
 11. The method of claim 10 wherein saidchelate is prepared by reducing a pertechnetrate in the presence of saidchelating agent.
 12. The method of claim 11 wherein said reduction iseffected utilizing stannous chloride as a reducing agent. .[.13. Amethod of preparing the chelate of claim 2 comprising reacting saidradio-isotope with said chelating agent..]..Iadd.
 14. A chelate oftechnetium-99m, cobalt-57, gallium-67, gallium-68, indium-111 orindium-113m and 5,7-diiodo-8-hydroxyquinoline. .Iaddend..Iadd.
 15. Amethod of external imaging which includes the intravenous administrationof a solution adapted for intravenous administration and containing thechelate of claim
 14. .Iaddend..Iadd.
 16. A method of preparing thechelate of claim 14 comprising reacting said radio-isotope with saidchelating agent. .Iaddend. .Iadd.
 17. A chelate of technetium-99m,cobalt-57, gallium-67, gallium-68, indium-111 or indium-113m and asubstituted iminodiacetic acid of the formula: .Iaddend. ##STR2##.Iadd.wherein R may be alkyl having up to 14 carbon atoms..Iaddend..Iadd.
 8. A composition comprising a mixture of thetechnetium-99m chelate of claim 17 and the stannous chelate of saidchelating agent. .Iaddend..Iadd.
 19. A composition comprising a mixtureof the technetium-99m chelate of claim 17, the stannous chelate of saidchelating agent and said chelating agent. .Iaddend..Iadd.
 20. A methodof external imaging which includes the intravenous administration of asolution adapted for intravenous administration containing the chelateof claim
 17. .Iaddend..Iadd.
 21. A method of preparing the chelate ofclaim 17 comprising reacting said radio-isotope with said chelatingagent. .Iaddend. .Iadd.
 22. A chelate of technetium-99m, cobalt-57,gallium-67, gallium-68, indium-111 or indium-113m andN-[N'-(2,6-dimethylphenyl) carbamoylmethyl] iminodiacetic acid..Iaddend. .Iadd.
 23. A chelate of technetium-99m, cobalt-57, gallium-67,gallium-68, indium-111 or indium-113m and methyliminodiacetic acid..Iaddend..Iadd.
 24. A chelate of technetium-99m, cobalt-57, gallium-67,gallium-68, indium-111 or indium-113m and 10-carboxydecyliminodiaceticacid. .Iaddend..Iadd.
 25. A chelate of technetium-99m, cobalt-57,galium-67, gallium-68, indium-111 or indium-113m and N-(o-bromobenzyl)iminodiacetic acid. .Iaddend..Iadd.
 26. A chelate of technetium-99m,cobalt-57, gallium-67, gallium-68, indium-111 or indium-113m and2,6-pyridinedicarboxylic acid. .Iaddend..Iadd.
 27. A chelate of claim 1wherein said radioisotope is technetium-99m. .Iaddend.